The present invention relates to methods and reagents for identifying compounds which inhibit proteolysis of polypeptides, especially membrane-associated polypeptides.
The amyloid precursor protein (APP) is an integral membrane protein located predominantly within intracellular vesicles. One of the well-characterized proteolytic processing pathways of APP results in the generation of a 40 or 42 amino acid peptide (A.beta.40-42). Extracellular deposition and plaque formation nucleated by these amyloid A.beta. peptides form a hallmark lesion in the brains of Alzheimer's disease patients [Selkoe, D. J. (1996) J. Biol. Chem. 271:18295-18298]. A.beta.40-42 peptides are generated from the specific and sequential cleavage of the membrane-bound APP by the .beta. and .gamma.-secretases [Selkoe, D. J. (1997) Science 275:630-631; Lamb, B. T. (1997) Nature Med. 3:28-29]. .beta.-secretase cleaves on the lumenal side of the ER and, together with the subsequent intrinsic membrane cleavage by .gamma.-secretase, determines the rate of A.beta.40-42 generation. Notably, the .gamma.-secretase cleavage of APP bears a striking similarity to that of the SREBP S2P cleavage and, except for Notch processing (discussed below), is the only other known example of specific proteolysis occurring within a membrane-spanning segment [Brown and Goldstein (1997) Cell 89:331-340; Sakai et al. (1996) Cell 85:1037-1046]. Recently, the APP .gamma.-secretase and SREBP S2P have been shown to be two distinct enzymes [Tomita et al. (1998) NeuroReport 9:911-913].
The Notch receptor family includes Notch in Drosophila, LIN-12 and GLP-1 in C. elegans, and mNotch1 and mNotch2 in mouse, among others [Artavanis-Tsakonas et al. (1995) Science 268:225-232]. During development, Notch mediates cell-cell communications required for a variety of cell fate decisions and for axon guidance. Notch family members are large, multidomain proteins that consist of a single transmembrane domain and large extracellular and intracellular domains. Proteolytic cleavage of Notch is believed to result in release of the intracellular domain, which translocates to the nucleus and associates with a DNA-binding subunit [Li, X, and Greenwald, I. (1998) Proc. Natl. Acad. Sci. USA 95:7109-7114; Thinakaran et al. (1996) Neuron 17:181-190; Podlisny et al. (1997) Neurobiol. Dis. 3:325-337; Capell et al. (1998) J. Biol. Chem. 273:3205-3211]. A processing step responsible for releasing the intracellular domain takes place in or near the transmembrane domain [Li and Greenwald (1998) supra]. Thus, Notch appears to undergo proteolytic events that resemble those involved in cleavage of APP, i.e., sequential hydrolysis by .beta. and .gamma.-secretases.
The regulation of hepatic cholesterol biosynthesis is central to the understanding of hypercholesterolemia as a risk factor for cardiovascular disease [Goldstein and Brown (1997) Nature 343:425-430]. A sterol-activated transcription factor, the sterol regulatory element binding protein (SREBP), specifically binds sterol regulatory elements (SREs) in the regulatory region of a number of coordinately regulated genes, including the promoters of the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [Briggs et al. (1993) Cell. 77:53-62; Wang et al. (1993) J. Biol. Chem. 268:14497-14504]. SREBP is maintained in an inactive state by its targeted localization to the endoplasmic reticulum (ER) membrane. Upon cholesterol deprivation, the amino-terminal portion of SREBP is specifically proteolyzed and liberated from its ER anchor in a two-step process [Wang et al. (1994) J. Biol. Chem. 271:10379-10384; Hua et al. (1996) J. Biol. Chem. 271:10379-10384; Sakai et al. (1996) supra]. This cleavage releases the amino-terminal segment of SREBP, allowing it to enter the nucleus, where it binds to enhancers and activates transcription of genes encoding the LDL receptor and multiple enzymes of cholesterol and fatty acid biosynthesis [Brown and Goldstein (1997) supra]. Sterols regulate initial proteolysis at site 1, which appears to be a prerequisite for site 2 hydrolysis, which occurs within the ER bilayer. The cDNA encoding the protease responsible for site 2 cleavage of the SREBPs (site 2 protease (S2P)) was isolated by complementation cloning and encodes a putative zinc metalloprotease with multiple transmembrane domains [Rawson et al. (1997) Mol. Cell. 1:47-57]. The site 1 protease (S1P), a membrane-bound subtilisin-related serine protease, has also recently been cloned and characterized [Espenshade et al. (1999) J. Biol. Chem. 274:22795-22804].
Due to the importance of membrane-associated proteolysis in gene expression, cellular differentiation and disease, considerable research effort has focused on membrane proteins, and particularly on identifying enzymes involved in their proteolytic pathways. Despite these efforts, until recently, only one of the several inferred proteolytic enzymes responsible for APP, SREBP and Notch maturation had been identified. Although at least three such proteases have now been cloned, specific and nontoxic inhibitors of these enzymes are not yet available. Thus, a need exists for an accurate, sensitive and economical high throughput screen to identify novel nontoxic inhibitors of proteolysis, especially membrane-associated proteolysis. In addition, due to the difficulty in isolating enzymes involved in lumenal and intrinsic membrane proteolysis, there exists a need for a simple and reliable technique which will enable tho large-scale screening of compounds for antiproteolytic activity without the need to first clone the target protease. The present invention fulfills these and other needs.